Synergistic preparation of rifampicin and penicillin g

ABSTRACT

Rifamycines or their semisynthetic derivatives, in combination with penicillines or cephalosporines, exhibit synergistic effects against pathogens.

United States Patent Konopka et al. lM8lch 13, 1973 1 SYNERGISTIC PREPARATION OF [58] Field of Search ..424/114 RIFAMPICIN AND PENICILLIN G [75] lnventors: Edward Alexander Konopka, Mur- [56] References Cited ray Hill; Justus Melchior Gelzer, OTHER PUBLICATIONS Summit, both of NJ.

Virchow et 3]., Dtsch. Med. Wschr., 92, Jg, N+48, [73] Assignee: Ciba-Geigy Corporation, Arclsley, D mber l967,pages 2217-2220.

1 NY. The Merck lndex, Merck & Co., 7th Edition, 1960, 22 Filed: Oct. 15,1970 Pages [21] Appl. No.: 81,112 Primary Examiner--Jcrome D. Goldberg Related US. Application Data Continuation-impart of Ser. No. 137,788, Feb. 24, 1970, abandoned, which is a continuation-in-part of Att0rney-Harry Goldsmith, Joseph G. Kolodny and Mario A. Monaco [57] ABSTRACT Rifamycines or their semisynthetic derivatives, in combination with penicillines or cephalosporines,-exhibit synergistic effects against pathogens.

1 Claim, No Drawings SYNERGISTIC PREPARATION OF RIFAMPICIN AND PENICILLIN G CROSS-REFERENCES TO RELATED APPLICATIONS This is a continuation-in-part of application Ser. No. 13,788, filed Feb. 24, 1970 now abandoned, which in turn is a continuation-impart of application Ser. No. 809,967, filed Mar. 24, 1969 now U.S. Pat. No. 3,644,616.

SUMMARY OF THE INVENTION The present invention concerns and has for its object the provision of new pharmaceutical or veterinary compositions, feedstuffs or feed additives comprising combinations of (l) rifamycines or their semisynthetic derivatives, with (2) other antibiotics, preferably penicillines'or cephalosporines, as well as of methods for the preparation and application of these products, Said compositions are useful antibiotics and the feed preparations useful growth promoters.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The rifamycines of the new compositions and feed preparations are known and represent especially the rifamycines B, SV, S, 0, AG or X, preferably their semisynthetic derivatives, e.g., rifamide, rifazine or advantageously rifampicin. Said component is described, inter alia, in 11 Farmaco, Ed. Sci. 16, 755 and 766 (1961), 21, 68 (1966) and 22, 307 (1967); J. Med. Chem. 7, 596 (1964), 8, 790 (1965) and 11, 936 (1968); Antimicrobial Agents and Chemotherapy (Am. Soc. Microbiol.) 1965, p. 765 or 1967, p. 699, the Report of the 5th Internatl. Congr. of Chemotherapy, 1967; French Pat. Nos. 1,434,532, 1,457,435 and 5518M, Belgian Pat. Nos. 654,209 and 685,886, South African Pat. No. 68/0903 and U.S. Pat. No. 3,349,082.

The antibiotics used are also known and are represented by the group of natural or "semisynthetic penicillines or cephalosporines, which are described, inter alia, in Erhart-Ruschig, Arzneimittel II, 1571 et seq. (Verlag Chemie, Weinheim 1968). Said book also describes the rifamycines on page 1517 et seq.

The compositions and feed preparations according to the invention contain an effective amount-of the rifamycines and other antibiotics in a ratio between about 1:10 and :1, preferably between about 1:5 and 5:1, especially between about 1:2 and 2:1, and the usual amount of conventional excipients or extenders, whereby the total amount of both antibiotics can be less than that used in the known preparations of the components. I

The antibacterial effects of the new combinations can be tested either in vitro or in vivo. For example, the

growth dynamics of Gram-negative or positive bacteria can be estimated in media which contain, besides the nutrients,

a. no antibiotic (control (b).

b. the rifamycines (A) or other antibiotics (b) alone,

0. both components (A B).

The single amounts of A and B used according to (0) can be half of those used according to (b). In vivo tests can be performed with various test animals, ad-

vantageously with mammals, such as mice, which are challenged with a lethal or sublethal amount of pathogens, whereupon the compositions of the invention are administered. Their effect can either be determined by the curing rate of infected animals or by the recovery of viable pathogens from their organism, for example, from particular organs, such as kidneys.

Surprisingly, it has been found that the growth dynamics of pathogens, more particularly of the antibiotically less tractable Gram-negative bacteria, such as the Aerobacter, Brucella, Escherichia, Klebsiella, Malleomyces, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella, Shiegella and Vibrio strains, as well as those of Gram-positive bacteria, such as Actinomyces, Clostridia, Corynebacteria, Diplococci, Mycobacteria, Staphylococci or Streptococci, are beneficially altered by the compositions of the invention. For example, said in vitro growth dynamics according to items (a), (b) and (c) can be depicted as follows:

Number n of surviving bacteria Time t 20 (hours) These functions f(n,t) depicting said growth dynamics indicate that the antibacterial effect of a combination according to the invention is bigger than that obtainable with the same amount of the components.

Accordingly, the new compositions and feed preparations are superior to those of the presently used components, since lesser doses can be applied. Moreover, a suppression of resistance development can be achieved. For example, pathogens attached with the rifamycines or their semisynthetic derivatives,- seem to be more sensitive to the penicillines or cephalosporines.

Particularly useful are pharmaceutical or veterinary compositions, as well as feedstuffs and feed additives, comprising an effective amount of l. a rifamycin selected from the group consisting of rifamycin (AG, B, O, S, SV or X), rifamide, rifampicin and rifazine or a therapeutically useful salt thereof and another antibiotic selected from the group consisting of penicillin BT, D, F, G, K. L, MV, N, O, S, V, W or X), ampicillin (A or B), ancillin, azidopenicillin, betacin, carbenicillin, chinacillin, cloxacillin, dicloxacillin, flucloxacillin, furfurylpenicillin, hetacillin, levopropylcillin, methicillin, nafcillin, oxacillin, phenoxyisopropylpenicillin, propicillin, thiphencillin; cephalosporin C, cephalexin, cephaloglycin, cephaloran, cephaloridine, cephalothin, cephazolin or hetasporin, or a therapeutically useful salt thereof.

Especially valuable are compositions and feed amount of l. rifamide, rifampicin or rifazine, or a therapeutically useful salt thereof and 2. penicillin (BT, G or V), ampicillin A, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, cephaloridine or cephalothin, or a therapeutically useful salt thereof.

Said compositions and feed preparations advantageously contain but one of the above antibiotics mentioned under items (1) and (2) but can contain more than one of each, for example, more than one of the antibiotics listed under item (2). The preferred proportions of the active ingredients of said compositions and feed preparations range between about 1:5 and 5:1, advantageously between about 1:2 and 2:1. In addition to the active ingredients, they contain the usual amount of conventional excipients or extenders.

The pharmaceutical or veterinary compositions according to the invention contain both of said antibiotics mentioned under (1) and 2) in about the same or a lesser amount than that used in conventional compositions of the components, in conjunction or admixture with excipients suitable for either enteral, parenteral or topical application. Preferred are tablets and gelatin capsules comprising the active ingredients together with (a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine, (b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol, for tablets also (0) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, if desired, (cl) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, enzymes of the binders or effervescent mixtures and/or (e) adsorbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories or ointments are advantageously fatty emulsions or suspensions. They may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. Said pharmaceutical compositions may also contain other therapeutically valuable substances. They are prepared according to conventional mixing, granulating or coating methods respectively and contain about 0.1 to 75 percent, preferably about 1 to 50 percent, of the active ingredients listed under l and (2).

The feedstuffs or additives for feed or drinking water contain both of said antibiotics also in about the same or a lesser amount as that used in conventional feedstuffs or additives of the components, which are intended to promote the growth and feed efficiency of domestic animals. Said feedstuffs or additives also contain the conventional extenders, diluents and/or nutrients, such as sucrose, glucose, molasses, fermentation residues, corn meal, ground and rolled oats, wheat shorts and middlings, meat scrap, oil cake, soybean and fish meal, alfalfa, clover or grass clippings and the like, mineral supplements, such as bone meal, calcium carbonate, iodized salt and the like, vitamins, such as vitamins A, B, C and D, and other suitable substances, such as preservants, e.g., benzoic acid. The feedstuffs contain the active ingredients advantageously in the dosage range, for example, between about 0.00001 and 0.01 percent, whereas the additives may consist of the pure substances, when used, for example, for the drinking water, but usually contain between about 1 and percent thereof.

The following examples illustrate the invention and are not to be construed as being limitations thereon. Temperatures are given in degrees centigrade and all parts wherever given are parts by weight.

EXAMPLE 1 Conventional test tubes are filled with 10 ml of conventional trypticase broth (0) alone, or such containing (d) rifampicin or the other antibiotics, or e) the combinations thereof, in a concentration between about 5-20 pg/ml.

Hereupon 0.1 ml ofa freshly grown and standardized stock culture of Escherichia coli is added to said 10 ml broth, so that it contains approximately 10 organisms per ml. After 20 hours incubation at 37, 1 m1 samples are removed and the number of viable cells therein determined according to the plate dilution method. It provides from a number of bacterial cells in a properly diluted specimen an equal number of visible colonies, which can be counted.

The following results are obtained:

White laboratory mice are infected by the i.p. application of a sufficient amount of the pathogens indicated in the following table, suspended in saline, which cause death of -100 percent of untreated control animals within 48 hours. About 30-60 minutes after infection, groups of 10 mice each are treated with the antibiotics shown in the table below, either alone or in combination, which antibiotics are administered only once in the form of aqueous solutions or suspensions, either orally (p.o.) or subcutaneously (s.c.). Said in vivo experiments are terminated at the 10th to 14th day after infection and the survivors counted. Most of the experiments were carried out twice and the average value estimated.

The following results were obtained:

Staphylococcus aureus A 0.05 p.o. 10 Procedure: B All powders are passed through a screen with an A B 0.05 0.2 40 I A 0.05 p.o. opening of 0.6 mm and mixed thoroughly. 0.5 ml hard 5 gelatine capsules are filled with 380 mg of said mixture, A+B 0.05-+0.1 A 01 5 5 using a capsule i ing mac ine. B 0.05 s.c. 0 A B 0.1 0.05 20 none 0 0 rifampicin (A) 0.01 pa. 10 EXAMPLE 6 cephaloridine (B) 0.05 s.c. O A B 0.05 0.05 60 10 A 01 P-- 20 Preparation of a poultry feed containing 0.005 per- R B 821 90 cent of the active ingredients:

EXAMPLE 3 Premix: i5 rifampicin g Groups of 10 white laboratory mice each are inamgicillin 2 5 con ectioners su ar g fected by the intravenous application of 10 cells of a soybean feed, solgvememacted 275 g penicillin G resistant strain of staphylococcus aureus in Total 375 g saline. Medication is applied orally or subcutaneously only once within 1 hour after infection. All surviving 20 animals are sacrificed on the 5th day after infection, Feed Formula: Pounds Corn meal 1,103.0 both kidneys of individual mice are removed asepti Soybean mealrwqb protein 660'0 cally and macerated in a tissue grinder. Appropriate Alf lf meal 300 dilutions are made with Soerensens buffer at pH=7 Dlcalc'um Phmphme Limestone meal 10.0 and the number of organisms present in said diluted 25 Salt kidney homogenate is determined by plating aliquots Sigi' fifii Protein 28-8 thereof on trypticase soy agar medium and incubating Dried whey it for 48 hours at 37. Manganese sulfate 0.5 Zinc oxide 0.3 In addition to the determination of the total number d p hi i 15 of viable cells in the diluted kidney homogenates, also 30 Vitamin premix T l 2 000 $9 the number of resistant cells within the total bacterial ma population is determined. This is done by counting the staph. aureus cells capable of growing in said trypticase I soy agar medium containing an additi l 5 f ii1 10 lb. of the vitamin composition contain: 16 million lin G per ml.

The following results'were obtained in recovering viable cells in the kidneys of individually treated and untreated mice:

LU. Vit. A, 1 million LU. Vit. D 5,000 1.U. Vit E acetate, 6 g Vit. K 6 mg Vit. B 3 g riboflavin, 30 g niacin, 5 g calcium pantothenate and 100 g ethoxyquin, made up to 10 lb. with corn meal. Procedure:

Rlfamplcln (A) 0.3 Penicillin G (B) 20 Controls mgJkg. p.o. mgJkg. s.c. mgJkg.

Total B Total ]3 Total Total 13-- nuniber resist. number resist. number resist. number resist.

2.2Xl0" 3.7X10 2.lX10 3.3)(10 7.6Xl0 1.0X10 1.7)(10 4.4X10 2.2Xl0 3.6)(10 6.0)(10 0 4.0)(10 1.5X10 7.4X10 1.5X10 2.8X1C- 2 8X10 3.6)(10 2.3Xl0 3.0X10' 1.2X10 7.5)(10 0 211x10 2 3X10 1.1)(10 4.4Xl0 2.3X10 6.1)(10 0 0 4.2Xl0' 8 0X10 1.3X10 7.0)(10 1.6Xl0 1.0X10' 1.4Xl0 0 1.2Xl0 1 6X10 8.0X10 1.3)(10 3.2X10 4.1)(10 4.2)(10 0 3.5X10 1 2X10 2.0)(10 6.1)(10' 1.3X10' 2.2)(10 0 0 4.2X10 3.4X10 2.1Xl0 7.8X10 1.8X10' l.lX10 2.3)(10 8.9X10

EXAMPLE 4 The antibiotics and sugar are mixed thoroughly,

Similar results as shown in Example 3 are obtained with strains of staph. aureus and E. coli, which are resistant to penicillins or cephalosporins, more particularly penicillin G and cephaloridine. After treatment with rifampicin, said resistant cells are again sensitive to said penicillins or cephalosporins.

EXAMPLE 5 Preparation of 1,000 capsules each containing 300 mg of the active ingredients:

Formula:

rifampicin 150 g ampicillin 150g talcum 36 g corn starch 24 3 magnesium stearate 16 g lactose 4 g Total 380 g screened through a sieve with 0.6 mm openings and blended with the soybean feed. The premix is then added to the feed in such amount as to obtain said concentration and the whole is homogenized in a horizontal drum mixer.

We claim: 

